Herbicide resistance gene, compositions and methods

Abstract

The present disclosure provides methods, recombinant DNA molecules, recombinant host cells containing the DNA molecules, and transgenic plant cells, plant tissue, seeds and plants which contain and express an herbicide resistant protoporphyrinogen oxidase such that they germinate from seed and grow in the presence of an amount of herbicide where the parent plant does not. Such plants are especially appropriate for use in agriculture or horticulture where herbicides are used to kill undesirable plants which might contaminate or compete with the transgenic plant of interest.

1. An isolated nucleic acid molecule comprising a coding sequence for an herbicide resistant protoporphyrinogen oxidase (PPO) wherein said PPO comprises deletion of one glycine residue corresponding to glycine-glycine at amino acids 210-211 of the PPO amino acid sequence of SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:28, or a sequence of one of the foregoing with from one to five amino acid substitution, deletion or insertion mutations, with the proviso that enzymatic activity and herbicide resistance are maintained. 
     
     
       2. The nucleic acid molecule of  claim 1 , wherein said herbicide resistant PPO is resistant to inhibition by lactofen, (±)-2-ethoxy-1-methyl-2-oxoethyl 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobenzoate; acifluorfen, 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobezoic acid; its methyl ester; or oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles, (e.g. oxidiazon, 3-{2,4-dichloro-5-(1-methylethylethoxy)phenyl}-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one), cyclic imides (e.g. S-23142, N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide), phenyl pyrazoles (e.g. TNPP-ethyl, ethyl 2-{1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy}propionate; M&B 39279), pyridine derivatives (e.g. LS 82-556), phenopylate and O-phenylpyrrolidino- and piperidinocarbamate analogs thereof, and 3-phenyluracils of formula I 
       
         
           
           
               
               
           
         
       
       wherein R 1  is methyl or NH 2 ; R 2  is C 1 -C 2 -haloalkyl; R 3  is hydrogen or halogen; R 4  is halogen or cyano; R 5  is hydrogen, cyano, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, C 3 -C 7 -cycloalkyl, C 3 -C 6 -alkenyl, C 3 -C 6 -alkynyl or benzyl which is unsubstituted or substituted by halogen or alkyl; and R 6 , R 7  independently of one another are hydrogen, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, C 3 -C 6 -alkenyl, C 3 -C 6 -alkynyl, C 3 -C 7 -cycloalkyl, C 3 -C 7 -cycloalkenyl, phenyl or benzyl, where each of the 8 abovementioned substituents is unsubstituted or may be substituted by 1 to 6 halogen atoms and/or by one, two or three groups selected from: OH, NH 2 , CN, CONH 2 , C 1 -C 4 -alkoxy, C 1 -C 4 -haloalkoxy, C 1 -C 4 -alkylthio, C 1 -C 4 -haloalkylthio, C 1 -C 4 -alkylsulfonyl, C 1 -C 4 -haloalkylsulfonyl, C 1 -C 4 -alkylamino, di(C 1 -C 4 -alkyl)amino, formyl, C 1 -C 4 -alkylcarbonyl, C 1 -C 4 -alkoxycarbonyl, C 1 -C 4 -alkylaminocarbonyl, di(C 1 -C 4 -alkyl)aminocarbonyl, C 3 -C 7 -cycloalkyl, phenyl and benzyl; or R 6 , R 7  together with the nitrogen atom form a 3-, 4-, 5-, 6- or 7-membered saturated or unsaturated nitrogen heterocycle which may be substituted by 1 to 6 methyl groups and which may contain 1 or 2 further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur as ring members. 
     
     
       3. The nucleic acid molecule of  claim 2 , wherein said PPO comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:26 or SEQ ID NO:30. 
     
     
       4. The nucleic acid molecule of  claim 3 , wherein said PPO comprises the amino acid sequence of SEQ ID NO:14. 
     
     
       5. The nucleic acid molecule of  claim 3 , wherein said PPO comprises the amino acid sequence of SEQ ID NO:26. 
     
     
       6. The nucleic acid molecule of  claim 3 , wherein said PPO comprises the amino acid sequence of SEQ ID NO:30. 
     
     
       7. The nucleic acid molecule of  claim 4 , wherein said herbicide resistant PPO sequence is encoded by SEQ ID NO:13 or SEQ ID NO:45. 
     
     
       8. The nucleic acid molecule of  claim 5 , wherein said PPO is encoded by SEQ ID NO:25. 
     
     
       9. The nucleic acid molecule of  claim 6 , wherein said PPO is encoded by SEQ ID NO:29. 
     
     
       10. The nucleic acid molecule of  claim 1 , further comprising a plant-expressible transcription regulatory sequence, said transcription regulatory sequence being operably linked to said coding sequence. 
     
     
       11. The nucleic acid molecule of  claim 10 , wherein said transcription regulatory sequence comprises a constitutive plant expressible promoter. 
     
     
       12. The nucleic acid of  claim 11 , wherein the plant expressible promoter is a  Arabidopsis thaliana  Act2 promoter, a Cauliflower mosaic virus 19 S promoter, a Cauliflower mosaic virus 35S promoter or a parsley ubiquitin promoter. 
     
     
       13. The nucleic acid of  claim 1 , further comprising a vector portion. 
     
     
       14. A method of using the nucleic acid molecule of  claim 1  to produce a transgenic plant cell, transgenic plant tissue or a transgenic plant which is resistant to an herbicide which specifically inhibits protoporphyrinogen oxidase, said method comprising the steps of introducing the nucleic acid molecule of  claim 1  into the plant cell, plant tissue or plant, selecting for the presence of the nucleic acid molecule to produce a transgenic plant cell or transgenic plant tissue, and optionally regenerating a plant from the transgenic plant cell or transgenic plant tissue, whereby a plant with an herbicide resistant phenotype is produced. 
     
     
       15. A transgenic plant containing and expressing the nucleic acid molecule of  claim 1 . 
     
     
       16. The transgenic plant of  claim 15 , wherein said plant is a dicotyledonous plant. 
     
     
       17. The transgenic plant of  claim 16 , wherein said plant is  Arabidopsis , cotton, sunflower, cabbage, broccoli, cauliflower, Brussels sprout, canola, bean, pea, soybean, citrus, tomato, potato, sweet potato, plum, peach, apple, pear, cherry, grape, rose, tobacco, alfalfa, trefoil, sugar beet, blackberry, raspberry, blueberry, marionberry, loganberry, apricot, eggplant, pepper, pumpkin, squash, gourd, an ornamental plant, aspen, poplar, maple, oak, sunflower, rose or tobacco. 
     
     
       18. The transgenic plant of  claim 15 , wherein said plant is a monocotyledonous plant. 
     
     
       19. The transgenic plant of  claim 18 , wherein said plant is wheat, corn, rice, sorghum, oat, wild rice, barley, millet or grass. 
     
     
       20. The transgenic plant of  claim 19 , wherein said plant is corn. 
     
     
       21. The transgenic progeny plant of the plant of any of  claims 15  to  20 . 
     
     
       22. Transgenic seed containing within its genetic complement the nucleic acid molecule of  claim 1 . 
     
     
       23. A method of controlling weeds comprising the step of planting transgenic plants comprising the nucleic acid molecule of  claim 1 ; and treating the plants with a PPO-inhibiting herbicide to which resistance is conferred by said molecule, whereby growth of weeds is controlled. 
     
     
       24. A method of selecting a transgenic plant cell containing and expressing the nucleic acid of  claim 1 , said method comprising the steps of growing said plant cell on medium comprising an amount of an inhibitor of porphoryinogen oxidase sufficient to prevent growth of a corresponding plant cell which does not contain and express said nucleic acid molecule. 
     
     
       25. An isolated protoporphyrinogen oxidase protein resistant to a PPO-inhbiting herbicide identified by the sequence given in SEQ ID NO:14, SEQ ID NO:26 or SEQ ID NO:30, or a sequence of the foregoing with from one to five amino acid substitutions, deletion or insertion mutations, provided that there is no insertion of a glycine residue after amino acid 210 of SEQ ID NO:14, SEQ ID NO:26 or SEQ ID NO:30. 
     
     
       26. The protoporphyrinogen oxidase of  claim 25 , wherein said protein consists of the sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:26 and SEQ ID NO:30.